Project Summary/Abstract Gap junctions are essential to normal tissue physiology, providing a direct intercellular pathway for cell to cell signaling and impulse conduction. Mutations in the genes of their subunit protein (connexins) have been linked to a number of human diseases. For instance, mutations in connexin40 (Cx40) have been associated with atrial fibrillation and other arrhythmias. Three Cx40 mutations (A96S, M163V and G38D) that correlate with atrial fibrillation have been previously shown to retain the ability to form conductive gap junction channels in vitro. Our recent publication demonstrates that two of the three have the same unitary conductance and voltage dependent behaviors as wild-type Cx40, while the third one has a significantly higher unitary conductance. All three mutants have altered permeability characteristics relative to wild-type channels. An increasing amount of evidence today indicates the important role of microRNAs in the pathogenesis and development of heart diseases. The data suggest another and as of yet untested possibility; these connexin mutants might affect cardiac conduction and pacing via altered permeability to microRNAs within the myocardium, which themselves target specific membrane K+ channels, IK1 (KCNJ2/Kir2.1) and IKr (HERG), that participate in conduction and pacemaking activities. It was recently reported that the microRNA miR-26 regulates IK1 expression and is down regulated in atrial fibrillation. miR-212 has also been shown to affect IK1 expression, whereas IKr expression is affected by miR-133. In this proposed study we will test if pacemaker activity can be modulated by connexin permeability to microRNAs/siRNAs that in turn regulate gene expression of specific membrane channels associated with pacemaker-like activity. In Aim 1 we will determine the permeability of gap junction channels formed by disease-linked mutations in Cx40 to miR-26, miR-212, miR-133, and siRNAs specific to IK1 and IKr, respectively, while simultaneously monitoring junctional conductance and cell to cell flux of microRNAs/siRNAs. In Aim 2 we will use a two-cell pacemaker model (a heterologous pair comprised of a source cell and a cardiac myocyte) to explore effects on pacemaker activity, by cellularly delivering microRNAs and siRNAs that target IK1 and IKr. Thereafter, in Aim3, we will analyze these results using our dynamic clamp model in order to add and subtract relevant currents associated with pacemaker activity to/from a single cell, a coupled pair, or two uncoupled cells paired electronically. In this proposed study, using a cellular biophysical approach, we seek to define if the permeability of gap junction channels might be an essential component in a process of dynamic gene expression that ultimately affects cellular and tissue functions in normal physiology and disease states.